Proteolytic activation of human factor V by thrombin results from the cleavage of three peptide bonds at Arg709, Arg1018, and Arg1545. In order to define the functional importance of these sites, mutants with isoleucine substitutions blocking thrombin cleavage at one, two, or all three activation sites were expressed in COS-7 cells. The wild type protein is activated approximately 10-fold by thrombin or Russell's viper venom (RVV-V). Thrombin cleavage at Arg709 alone did not result in an increase in procoagulant activity. Cleavage at both Arg709 and Arg1018 resulted in an approximately 3.4-fold increase in activity. Cleavage at these sites was required for rapid cleavage by thrombin at Arg1545, however, which resulted in maximal activation of the factor V molecule. In contrast, isolated cleavage at Arg1545 by RVV-V was sufficient for efficient and complete activation of factor V. The effect of isoleucine substitutions at one or both thrombin cleavage sites in a B-domain deletion mutant lacking amino acids 811-1491 was also investigated. The specific activity of all four mutants was approximately 30% compared to thrombin activated factor V, indicating that these isoleucine substitutions do not drastically alter the structure of the protein and that cleavage at these sites is not required for the expression of partial procoagulant activity.